The present invention is directed to isolation and identification of plant proanthocyanidin extracts. These extracts can be obtained from any of a variety of proanthocyanidin-containing plants including members of the plant families Ericaceae, Rosaceae, Pinaceae and Vitaceae, and preferably are from cranberry plants (especially Vaccinium macrocarpon), other Vaccinium spp. and grapes (Vitus spp.) These extracts are useful for prevention and treatment of urinary tract infections caused by P-type fimbriated Escherichia coli as well as other uses. Extracts containing proanthocyanidins with A-type interflavanoind bonds have been found to have potent bioactivity for inhibiting adherence of P-type E. coli. 
Millions of women each year are diagnosed with cystitis (bladder infections) and pyelonephritis (kidney infections). Countless numbers of dogs and cats also suffer from chronic urinary infections and die from renal infections. E. coli bacteria is the most common pathogen associated with these infections, causing over 80% of urinary tract infections. Over 30% of women suffer recurrent infections within a 6 to 12-month period and are forced to resort to extended use of antibiotics to treat these infections. Recurrent use of antibiotics can lead to pathogen resistance and result in deleterious side effects and toxicity reactions. Consequently there exists a need for safe alternative medications (e.g., non-antibiotics) that can be used to prevent or treat urinary tract infections in both animals and humans.
Cranberry juice has been shown to reduce bacteriuria associated with urinary tract infections in humans (Avorn et al., 1994, J. Am. Med. Soc. 271:751-754). The effect appears to be due to the ability of certain cranberry compounds to inhibit adhesion of type 1 (implicated in bladder infections) and P-type (implicated in kidney infections) E. coli bacterial phenotypes to human bladder epithelial cells (Sobota, 1984, J. Urol. 131:1013-1016; Schmidt and Sobota, 1988, Microbios. 55:173-181; Zafriri et al., 1989, Antimicrob. Agents Chemo. 33:92-98). Zafriri et al. (1989) reported that fructose was responsible for the inhibition of type 1 E. coli to uroepithelial cells. Zafriri et al. also reported that cranberry juice contained a non-dialyzable substance (or substances) which inhibited binding of P-type E. coli but failed to define the chemical nature of this inhibitor. Cranberry juice has also been shown to cause immediate inhibition of agglutination as well as loss of fimbriae after long-term exposure of bacteria to the juice (Ahuja et al., 1998, J. Urol. 159:559-562).
A partially-purified anti-adherence activity from cranberry has also been described (U.S. Pat. Nos. 5,474,774; 5,525,341; and 5,646,178, all to Walker et al.). This activity was obtained using acidified alcohol as an extraction solvent with whole cranberry fruit followed by separation of the activity from monomer and dimer sugars by precipitation with a metal acetate or sulfate. Upon further manipulation, the reported activity consisted of a fraction enriched for polyphenol and flavonoid compounds that contained as much as 10% anthocyanins. The specificity of this anti-adherence activity for type 1 or P-type E. coli was not determined.
Walker et al. (WO 96/30033; and U.S. Pat. Nos. 5,646,178 and 5,650,432) described a series of proanthocyanidin monomers, dimers, polymers, flavonoid derivatives thereof and related compounds purported to have the ability to interfere with bacterial adherence to a surface. The dimers and polymers of Walker were limited to compounds having B-type interflavanoid linkages. However, Walker failed to provide any experimental data correlating biological activity with a specifically-identified compound. The Walker method involved alkalinizing a plant material homogenate to a pH greater than 10, a treatment which causes degradation of proanthocyanidins, and precipitating the polyphenolic compounds (together with other materials) by addition of alcohol. This precipitate contained the proposed anti-adherence activity and was further fractionated to yield the purified active compound. Using this process with an aqueous solution of commercially-available Ocean Spray cranberry powder, Walker reported obtaining a single active compound and partially characterized the compound but failed to provide a complete (or any) chemical structure for this compound. Walker also failed to characterize the biological activity of this compound with respect to inhibition of adherence of type 1 or P-type E. coli. In fact, the Walker assay methods could not distinguish between these two biological activities.
Thus, prior to the present invention, there had been no identification of the class of bioactive compounds that inhibit P-type E. coli from adhering to surfaces such as uroepithelial cells. In accordance with the invention, it has been discovered that extracted mixtures of proanthocyanidins and purified proanthocyanidins are the bioactive compounds present in cranberry and other plants that possess anti-adherence activity against P-type E. coli. 
A large variety of plants are known to contain proanthocyanidins and methods for isolating small amounts of proanthocyanidins from several different plant species have been reported. Various purification methods for proanthocyanidins from plant material have been described by Thompson et al. (1972, J. Chem. Soc., Perkins Trans. I. 11:1387); Jones et al. (1976, Phytochem. 15:1407-1409); Wang et al. (1978, J. Food Sci. 43:1402-1404); Czochanska et al. (1980, J. Chem. Soc., Perkin Trans. I:2278-2286); Foo and Porter (1981, J. Sci. Food Agric. 32:711-716); Marwan and Nagel (1986a, J. Food Sci. 51:1009-1013); Marwan and Nagel (1986b, J. Food Sci. 51:1069-1070); Morimoto et al. (1988, Chem. Pharm. Bull. 36:33-38); Devlin, U.S. Pat. No. 4,309,207; and Bomser et al. (1996, Planta Med. 62:212-216).
Accordingly, the present invention identifies proanthocyanidins as the compounds that mediate inhibition of adherence of P-type E. coli to cellular surfaces and further provides an improved method of obtaining substantially pure mixtures of proanthocyanidins as well as individual proanthocyanidin compounds from the plant material of cranberries and other plants known to contain proanthocyanidins. These proanthocyanidin mixtures and individual proanthocyanidin compounds are thus useful in the prevention and/or therapeutic treatment of urinary tract infections, particularly those infections mediated by P-type E. coli or other microorganisms that contain structurally-related fimbriae or molecules involved in microbial adherence.
There also exists a need for an inexpensive and rapid test to identify the type of urinary tract infection that a person has contracted. Current clinical diagnosis is based on counting the number of bacteria present in a patient""s urine without attempting to distinguish the bacterial strain as type 1 E. coli, P-type E. coli or a mixture of both. Since kidney infections (associated more with P-type E. coli) are usually more serious than bladder infections (associated more with type 1 E. coli) and require different treatment regimes, having a test to distinguish between these phenotypic strain variations would be beneficial. Thus, an inexpensive, rapid diagnostic kit to diagnose P-type infection would allow patients to receive the proper treatment in a more timely manner and avoid the use of ineffective medications. Accordingly, the invention also addresses this problem by providing proanthocyanidin mixtures which specifically bind to P-type E. coli as receptor analogs or prevent P-type fimbrial induction for assay kits designed to aid in the clinical diagnosis of pyelonephritis-type infections.
The present invention is directed to proanthocyanidin extracts substantially free of anthocyanins and flavonols. These extracts are also shown to be free of hydrolyzable tannins, alkaloids, lipids, carbohydrates, simple sugars, protein and amino acids, alcohols and organic acids by chemical reagent testing. These proanthocyanidins extracts are capable of inhibiting agglutination reactions of P-type E. coli but not type 1 E. coli. In particular, those extracts containing proanthocyanidins with at least one A-type interflavanoid bond have been found to be more bioactive in agglutination reactions than proanthocyanidins linked only by B-type interflavanoid bonds.
The invention also provides a method of obtaining these proanthocyanidin extracts from the plant material from any of a variety of proanthocyanidin-containing plants. The preferred plants are in the Ericaceae, Rosaceae, Pinaceae and Vitaceae families, particularly the Vaccinium species of the Ericaceae family, and the Vitis species of the Vitaceae family, and most preferably from cranberries (especially from V. macrocarpon). The method is applicable for extracting the proanthocyanidins from any portion of the plant including leaves, ripe fruit and unripe fruit. The method of the invention causes substantially less oxidative damage and/or structural degradation to the proanthocyanidins, i.e., is less harsh, and provides improved yields of purified proanthocyanidins relative to known methods.
More particularly, the method of the invention is directed to preparing a proanthocyanidin extract from a plant by (a) homogenizing plant material in an aqueous extraction solvent comprising at least about 10% water but no more than about 30% water, about 10% to about 70% acetone, about 5% to about 60% methanol and about 0.05% to about 1% ascorbic acid; and subjecting that extract to further purification to allow recovery of a substantially purified proanthocyanidin extract which is capable of inhibiting agglutination of P-type E. coli but incapable of inhibiting agglutination of type 1 E. coli. 
Hence, a preferred embodiment of the invention relates to a method of preparing a proanthocyanidin extract from a Vaccinium species which comprises:
(a) homogenizing Vaccinium plant material in an aqueous extraction solvent comprising at least about 10% water but no more than about 30% water, about 10% to about 70% acetone, about 5% to about 60% methanol and about 0.05% to about 1% ascorbic acid to prepare a first extract;
(b) clarifying and obtaining the supernatant from the first extract;
(c) removing the solvent from the supernatant and resuspending the residue in distilled water;
(d) subjecting the resuspended residue solution to further purification by either
(i) applying the residue solution to reverse-phase lipophilic chromatography material equilibrated in distilled water and successively washing the lipophilic chromatography material with distilled water to remove sugars, with about 15% aqueous methanol to remove acids and with about 100% acidified methanol to elute polyphenolic compounds, and removing solvent from the polyphenolic fraction to obtain a first dried polyphenolic fraction, or
(ii) extracting the residue solution with a non-polar extraction solvent, recovering the aqueous phase and removing solvent therefrom to obtain a second dried fraction;
(e) suspending the first or second dried fraction in about 50% aqueous ethanol, applying that solution to mixed hydrophilic-lipophilic chromatography material equilibrated in about 50% aqueous ethanol, and washing the mixed hydrophilic-lipophilic chromatography material with about 50% aqueous ethanol to remove non-proanthocyanidin polyphenolic compounds; and
(f) eluting the mixed hydrophilic-lipophilic chromatography material with about 70% aqueous acetone to obtain the proanthocyanidin extract. This proanthocyanidin extract inhibits the agglutination of P-type E. coli but does not inhibit agglutination of type 1 E. coli. 
The proanthocyanidin extract can be further fractionated using HPLC or other techniques to identify and characterize specific proanthocyanidin compounds that have anti-adherence activity against P-type E. coli or other microorganisms. Such compounds include (a) proanthocyanidin compounds having an average of at least four to about twelve epicatechin flavanoid units, each linked to the next by a B-type interflavanoid bond between C4 and C8 or between C4 and C6 and (b) proanthocyanidin compounds having an average of at least four to about seven epicatechin flavanoid units, wherein at least two of the units are linked together by an A-type double interflavanoid bond between C4 and C8 and between C2 and the oxygen of C7 and the remaining units are linked to each other by a B-type interflavanoid bond between C4 and C8 or between C4 and C6.
Another aspect of the invention relates to methods of preventing or treating urogenital infections in a mammal by administering a proanthocyanidin composition comprising the proanthocyanidin extract, a proanthocyanidin compound, a proanthocyanidin polymer or a mixture thereof, to the mammal in an amount and for a time sufficient to prevent, reduce or eliminate the symptoms associated with such infections and thereby lead to an amelioration or curing of the infection. Preferably the mammal undergoing treatment is a human, but the method is also applicable to animals, especially domesticated animals such as cats and dogs and livestock animals such as cattle.
As used herein a xe2x80x9cproanthocyanidin compositionxe2x80x9d comprises a proanthocyanidin extract of the invention, a proanthocyanidin compound of the invention, a proanthocyanidin polymer or a mixture thereof. The proanthocyanidin composition can be provided as a pharmaceutical composition, e.g., in pill form, as a food additive, e.g. for a beverage, or as a food composition. When a proanthocyanidin composition of the invention is provided in a cranberry juice beverage, the proanthocyanidins can enhance the cranberry juice""s known benefits for preventing and treating urinary tract infections.
Hence, pharmaceutical compositions are provided which comprise a proanthocyanidin composition, including pharmaceutically-acceptable salts of any of the proanthocyanidin compounds or polymers, with a pharmaceutically acceptable carrier. In some instances, it may be preferable to provide the therapeutic dosage in the form of a food additive in a beverage such as a cranberry juice-based beverage containing additional proanthocyanidins. The invention also provides food compositions comprising a proanthocyanidin composition, including pharmaceutically-acceptable salts of the compounds or polymers, mixed with a consumable carrier. Consumable carriers include, but are not limited to, livestock feed, domestic animal feed and consumable food products, especially a cranberry-containing food products. These food compositions are also useful to prevent or treat urinary tract infections.
In another embodiment, the proanthocyanidin composition of the invention can be used to reduce the pathogenesis of P-type E. coli found in the digestive tracts of cattle. Such a method may be useful in decreasing contamination of ground meat prepared from such cattle.
Yet another aspect of the invention relates to a method of detecting P-type E. coli in a body fluid, especially in urine, and use of that method in diagnosis of pyelonephritis or other urinary tract infection associated with P-type E. coli. A kit for detecting P-type E. coli is also provided.
A still further aspect of the invention is directed to methods of reducing the incidence of infection after surgery, treating topical wounds and acne, and preventing or eliminating oral infections using the proanthocyanidin composition of the invention.
Further still, the proanthocyanidin composition of the invention can also be used as a food additive to confer protection against E. coli present in certain food products such as ground meat and unpasteurized juices, or as a feed additive to reduce the pathogenesis of P-type E. coli found in the digestive tracts of animals, especially cattle destined for slaughter. When used as a food additive for ground meat, it is preferable to add the proanthocyanidin composition to the meat preparations before or during grinding thereof